Combined analysis of the microbiome, metabolome and transcriptome of silkie chickens in response to avian pathogenic E. coli (APEC).

Avian colibacillosis is a bacterial disease caused by avian pathogenic Escherichia coli (APEC) that results in great losses in the poultry industry every year. Individual Silkie chickens of the same breed that are given the same feed in the same feeding conditions have different levels of resistance or susceptibility to APEC. Differences in gut microbes, gut metabolites, and gene expression in the spleen of APEC-resistant and APEC-susceptible chickens were compared, and multiple omics associations were analyzed to explore the mechanism of resistance to APEC in Silkie chickens. Compared with those in the APEC-susceptible group, the APEC-resistant group showed significantly increased abundances of many gut microorganisms, including Bacillus, Thermoactinomyces, Arthrobacter, and Ureibacillus, which were positively correlated with norvaline, l-arginine, and valyl-glycine levels. Intestinal tryptophan, indole, and indole derivative-related differentially abundant metabolites played an active role in combatting APEC infection. In the spleen, "response to stimulus" was the most significantly enriched GO term, and "cytokine‒cytokine receptor interaction" was the most significantly enriched KEGG pathway. The arginine biosynthesis and PPAR signaling pathways were the KEGG pathways that were significantly enriched with differentially abundant metabolites and differentially expressed genes. This study provides new insight into the prevention and treatment of APEC infection in Silkie chickens and lays a foundation to study the mechanism of APEC infection in poultry.

CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression.

Non-coding RNAs (ncRNAs) induced competing endogenous RNAs (ceRNA) play crucial roles in various biological process by regulating target gene expression. However, the studies of ceRNA networks in the regulation of ovarian ovulation processing of chicken remains deficient compared to that in mammals. Our present study revealed that circEML1 was differential expressed in hen's ovarian tissues at different ages (15 W/20 W/30 W/68 W) and identified as a loop structure from EML1 pre-mRNA, which promoted the expressions of CYP19A1/StAR and E2/P4 secretion in follicular granulosa cells (GCs). Furthermore, circEML1 could serve as a sponge of gga-miR-449a and also found that IGF2BP3 was targeted by gga-miR-449a to co-participate in the steroidogenesis, which possibly act the regulatory role via mTOR/p38MAPK pathways. Meanwhile, in the rescue experiment, gga-miR-449a could reverse the promoting role of circEML1 to IGF2BP3 and steroidogenesis. Eventually, this study suggested that circEML1/gga-miR-449a/IGF2BP3 axis exerted an important role in the steroidogenesis in GCs of chicken.

Genome-wide association study of 17 serum biochemical indicators in a chicken F2 resource population.

BACKGROUND: Serum biochemical indicators are often regarded as direct reflections of animal metabolism and health. The molecular mechanisms underlying serum biochemical indicators metabolism of chicken (Gallus Gallus) have not been elucidated. Herein, we performed a genome-wide association study (GWAS) to identify the variation associated with serum biochemical indicators. The aim of this research was to broaden the understanding of the serum biochemical indicators in chickens. RESULTS: A GWAS of serum biochemical indicators was carried out on 734 samples from an F2 Gushi× Anka chicken population. All chickens were genotyped by sequencing, 734 chickens and 321,314 variants were obtained after quality control. Based on these variants, a total of 236 single-nucleotide polymorphisms (SNPs) on 9 chicken chromosomes (GGAs) were identified to be significantly (-log10(P) > 5.72) associated with eight of seventeen serum biochemical indicators. Ten novel quantitative trait locis (QTLs) were identified for the 8 serum biochemical indicator traits of the F2 population. Literature mining revealed that the ALPL, BCHE, GGT2/GGT5 genes at loci GGA24, GGA9 and GGA15 might affect the alkaline phosphatase (AKP), cholinesterase (CHE) and γ-glutamyl transpeptidase (GGT) traits, respectively. CONCLUSION: The findings of the present study may contribute to a better understanding of the molecular mechanisms of chicken serum biochemical indicator regulation and provide a theoretical basis for chicken breeding programs.

Genetic architecture and key regulatory genes of fatty acid composition in Gushi chicken breast muscle determined by GWAS and WGCNA.

BACKGROUND: Fatty acids composition in poultry muscle is directly related to its tenderness, flavour, and juiciness, whereas its genetic mechanisms have not been elucidated. In this study, the genetic structure and key regulatory genes of the breast muscle fatty acid composition of local Chinese chicken, Gushi-Anka F2 resource population by integrating genome-wide association study (GWAS) and weighted gene co-expression network analysis (WGCNA) strategies. GWAS was performed based on 323,306 single nucleotide polymorphisms (SNPs) obtained by genotyping by sequencing (GBS) method and 721 chickens from the Gushi-Anka F2 resource population with highly variable fatty acid composition traits in the breast muscle. And then, according to the transcriptome data of the candidate genes that were obtained and phenotypic data of fatty acid composition traits in breast muscle of Gushi chickens at 14, 22, and 30 weeks of age, we conducted a WGCNA. RESULTS: A total of 128 suggestive significantly associated SNPs for 11 fatty acid composition traits were identified and mapped on chromosomes (Chr) 2, 3, 4, 5, 13, 17, 21, and 27. Of these, the two most significant SNPs were Chr13:5,100,140 (P = 4.56423e-10) and Chr13:5,100,173 (P = 4.56423e-10), which explained 5.6% of the phenotypic variation in polyunsaturated fatty acids (PUFA). In addition, six fatty acid composition traits, including C20:1, C22:6, saturated fatty acid (SFA), unsaturated fatty acids (UFA), PUFA, and average chain length (ACL), were located in the same QTL intervals on Chr13. We obtained 505 genes by scanning the linkage disequilibrium (LD) regions of all significant SNPs and performed a WGCNA based on the transcriptome data of the above 505 genes. Combining two strategies, 9 hub genes (ENO1, ADH1, ASAH1, ADH1C, PIK3CD, WISP1, AKT1, PANK3, and C1QTNF2) were finally identified, which could be the potential candidate genes regulating fatty acid composition traits in chicken breast muscle. CONCLUSION: The results of this study deepen our understanding of the genetic mechanisms underlying the regulation of fatty acid composition traits, which is helpful in the design of breeding strategies for the subsequent improvement of fatty acid composition in poultry muscle.

miR-128-3p inhibits intramuscular adipocytes differentiation in chickens by downregulating FDPS.

BACKGROUND: Intramuscular fat (IMF) content is the major indicator for evaluating chicken meat quality due to its positive correlation with tenderness, juiciness, and flavor. An increasing number of studies are focusing on the functions of microRNAs (miRNAs) in intramuscular adipocyte differentiation. However, little is known about the association of miR-128-3p with intramuscular adipocyte differentiation. Our previous RNA-seq results indicated that miR-128-3p was differentially expressed at different periods in chicken intramuscular adipocytes, revealing a possible association with intramuscular adipogenesis. The purpose of this research was to investigate the biological functions and regulatory mechanism of miR-128-3p in chicken intramuscular adipogenesis. RESULTS: The results of a series of assays confirmed that miR-128-3p could promote the proliferation and inhibit the differentiation of intramuscular adipocytes. A total of 223 and 1,050 differentially expressed genes (DEGs) were identified in the mimic treatment group and inhibitor treatment group, respectively, compared with the control group. Functional enrichment analysis revealed that the DEGs were involved in lipid metabolism-related pathways, such as the MAPK and TGF-ß signaling pathways. Furthermore, target gene prediction analysis showed that miR-128-3p can target many of the DEGs, such as FDPS, GGT5, TMEM37, and ASL2. The luciferase assay results showed that miR-128-3p targeted the 3' UTR of FDPS. The results of subsequent functional assays demonstrated that miR-128-3p acted as an inhibitor of intramuscular adipocyte differentiation by targeting FDPS. CONCLUSION: miR-128-3p inhibits chicken intramuscular adipocyte differentiation by downregulating FDPS. Our findings provide a theoretical basis for the study of lipid metabolism and reveal a potential target for molecular breeding to improve meat quality.

Comparative analyses of dynamic transcriptome profiles highlight key response genes and dominant isoforms for muscle development and growth in chicken.

BACKGROUND: Modern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler. RESULTS: Two time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2. TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts. CONCLUSIONS: For the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

Transcriptome identification and characterization of long non-coding RNAs in the ovary of hens at four stages.

Long non-coding RNAs (lncRNAs) play important roles in transcriptional and post-transcriptional regulation. LncRNAs, which are defined as non-coding RNAs more than 200 bp in length, are involved in key biological processes, such as cell proliferation and differentiation, epigenetic regulation, and gene transcriptional translation. Recent studies have shown that lncRNAs also play major regulatory roles in the reproduction of mammals. However, knowledge of the roles of lncRNAs in the chicken ovary lacking. In this study, we performed RNA-seq analyses of ovarian tissue from Hy-Line brown laying hens at four physiological stages [15, 20, 30, and 68 weeks of age (W)]. We identified 657 lncRNA transcripts that were differentially expressed during ovarian development, the number of down-regulated lncRNAs was higher than the number of up-regulated lncRNAs during development. We predicted the cis and trans target genes of the DE lncRNAs and constructed a lncRNA-mRNA interaction network, which indicated that the DE genes (DEGs) and the target genes of the DE lncRNAs are mainly involved in signaling pathways associated with ovarian development, including oocyte meiosis, calcium signaling pathways, ECM-receptor interactions, and ribosome and focal adhesion. Overall, we found that twelve lncRNAs were strongly involved in ovarian development: LNC_013443, LNC_001029, LNC_005713, LNC_016762, ENSGALT00000101857, LNC_003913, LNC_013692, LNC_012219, LNC_004140, ENSGALT00000096941, LNC_009356, and ENSGALT00000098716. In summary, our study utilized RNA-seq analysis of hen ovaries to explore key lncRNAs involved in ovarian development and function. Furthermore, the comprehensive analysis identified the target genes of these lncRNAs providing a better understanding of the mechanisms underlying ovarian development in hens and a theoretical basis for further research.

Identification of an Acinetobacter pittii acyltransferase involved in transformation of deoxynivalenol to 3-acetyl-deoxynivalenol by transcriptomic analysis.

Deoxynivalenol (DON), a mycotoxin primarily produced by Fusarium graminearum (F. graminearum), is widely present in food and feed, posing great hazards to human and livestock health. In this study, a strain of Acinetobacter pittii (A. pittii) S12 capable of degrading DON was isolated from soil samples and identified through morphological characterization, biochemistry analysis, and 16 S rRNA gene sequencing. The results of HPLC-MS indicated that the degradation products underwent a conversion from [M-H]- to [M+CH3CO], with concomitant transformation of the hydroxyl group into an acetyl moiety. Based on transcriptome sequencing analysis, the acyltransferase encoded by DLK06_RS13370 was predicted to be the pivotal gene responsible for DON biotransformation. The result of molecular docking analysis suggest a high affinity between the enzyme and DON. The recombinant protein encoded by DLK06_RS13370 was expressed in Escherichia coli (E. coli) and demonstrated the capacity to catalyze the conversion of DON into 3-Acetyl-deoxynivalenol (3-ADON), as confirmed by HPLC analysis. In conclusion, our findings confirm that the acyltransferase encoded by DLK06-RS13370 is responsible for the acetylation of DON. This sheds light on the co-occurrence of DON and its acetyl-derivatives in wheat-based products. DATA AVAILABILITY: Not applicable.

Genome-wide association study identifies SNPs for growth performance and serum indicators in Valgus-varus deformity broilers (Gallus gallus) using ddGBS sequencing.

BACKGROUND: Valgus-varus deformity (VVD) is a lateral or middle deviation of the tibiotarsus or tarsometatarsus, which is associated with compromised growth, worse bone quality and abnormal changes in serum indicators in broilers. To investigate the genetic basis of VVD, a genome wide association study (GWAS) was performed to identify candidate genes and pathways that are responsible for VVD leg disease, serum indicators and growth performance in broilers. RESULTS: In total, VVD phenotype, seven serum indicators and three growth traits were measured for 126 VVD broilers (case group) and 122 sound broilers (control group) based on a high throughput genome wide genotyping-by-sequencing (GBS) method. After quality control 233 samples (113 sound broilers and 120 VVD birds) and 256,599 single nucleotide polymorphisms (SNPs) markers were used for further analysis. As a result, a total of 5 SNPs were detected suggestively significantly associated with VVD and 70 candidate genes were identified that included or adjacent to these significant SNPs. In addition, 43 SNPs located on Chr24 (0.22 Mb - 1.79 Mb) were genome-wide significantly associated with serum alkaline phosphatase (ALP) and 38 candidate genes were identified. Functional enrichment analysis showed that these genes are involved in two Gene Ontology (GO) terms related to bone development (cartilage development and cartilage condensation) and two pathways related to skeletal development (Toll-like receptor signaling pathway and p53 signaling pathway). BARX2 (BARX homeobox 2) and Panx3 (Pannexin 3) related to skeleton diseases and bone quality were obtained according to functional analysis. According to the integration of GWAS with transcriptome analysis, HYLS1 (HYLS1 centriolar and ciliogenesis associated) was an important susceptibility gene. CONCLUSIONS: The results provide some reference for understanding the relationship between metabolic mechanism of ALP and pathogenesis of VVD, which will provide a theoretical basis for disease-resistant breeding of chicken leg soundness.

ELOVL gene family plays a virtual role in response to breeding selection and lipid deposition in different tissues in chicken (Gallus gallus).

BACKGROUND: Elongases of very long chain fatty acids (ELOVLs), a family of first rate-limiting enzymes in the synthesis of long-chain fatty acids, play an essential role in the biosynthesis of complex lipids. Disrupting any of ELOVLs affects normal growth and development in mammals. Genetic variations in ELOVLs are associated with backfat or intramuscular fatty acid composition in livestock. However, the effects of ELOVL gene family on breeding selection and lipid deposition in different tissues are still unknown in chickens. RESULTS: Genetic variation patterns and genetic associations analysis showed that the genetic variations of ELOVL genes were contributed to breeding selection of commercial varieties in chicken, and 14 SNPs in ELOVL2-6 were associated with body weight, carcass or fat deposition traits. Especially, one SNP rs17631638T > C in the promoter of ELOVL3 was associated with intramuscular fat content (IMF), and its allele frequency was significantly higher in native and layer breeds compared to that in commercial broiler breeds. Quantitative real-time PCR (qRT-PCR) determined that the ELOVL3 expressions in pectoralis were affected by the genotypes of rs17631638T > C. In addition, the transcription levels of ELOVL genes except ELOVL5 were regulated by estrogen in chicken liver and hypothalamus with different regulatory pathways. The expression levels of ELOVL1-6 in hypothalamus, liver, abdominal fat and pectoralis were correlated with abdominal fat weight, abdominal fat percentage, liver lipid content and IMF. Noteworthily, expression of ELOVL3 in pectoralis was highly positively correlated with IMF and glycerophospholipid molecules, including phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol and phospholipids inositol, rich in ω-3 and ω-6 long-chain unsaturated fatty acids, suggesting ELOVL3 could contribute to intramuscular fat deposition by increasing the proportion of long-chain unsaturated glycerophospholipid molecules in pectoralis. CONCLUSIONS: In summary, we demonstrated the genetic contribution of ELOVL gene family to breeding selection for specialized varieties, and revealed the expression regulation of ELOVL genes and their potential roles in regulating lipid deposition in different tissues. This study provides new insights into understanding the functions of ELOVL family on avian growth and lipid deposition in different tissues and the genetic variation in ELOVL3 may aid the marker-assisted selection of meat quality in chicken.

Weighted gene co-expression network indicates that the DYNLL2 is an important regulator of chicken breast muscle development and is regulated by miR-148a-3p.

BACKGROUND: The characteristics of muscle fibers determine the growth and meat quality of poultry. In this study, we performed a weighted gene co-expression network analysis (WGCNA) on the muscle fiber characteristics and transcriptome profile of the breast muscle tissue of Gushi chicken at 6, 14, 22, and 30 weeks. RESULTS: A total of 27 coexpressed biological functional modules were identified, of which the midnight blue module had the strongest correlation with muscle fiber and diameter. In addition, 7 hub genes were found from the midnight blue module, including LC8 dynein light chain 2 (DYNLL2). Combined with miRNA transcriptome data, miR-148a-3p was found to be a potential target miRNA of DYNLL2. Experiments on chicken primary myoblasts (CPMs) demonstrated that miR-148a-3p promotes the expression of myosin heavy chain (MYHC) protein by targeting DYNLL2, proving that it can promote differentiation of myoblasts. CONCLUSIONS: This study proved that the hub gene DYNLL2 and its target miR-148-3p are important regulators in chicken myogenesis. These results provide novel insights for understanding the molecular regulation mechanisms related to the development of chicken breast muscle.

Genetic effect of an InDel in the promoter region of the NUDT15 and its effect on myoblast proliferation in chickens.

BACKGROUND: Molecular breeding accelerates the speed of animal breeding. Screening molecular markers that can affect economic traits through genome-wide association studies (GWAS) can provide a theoretical basis for molecular breeding. At present, a large number of molecular markers have been screened in poultry research, but few reports on how molecular markers affect economic traits exist. It is particularly important to reveal the action mechanisms of molecular markers, which can provide more accurate information for molecular breeding. RESULTS: The aim of this study was to investigate the relationships between two indels (NUDT15-indel-2777 and NUDT15-indel-1673) in the promoter region of NUDT15 and growth and carcass traits in chickens and to explore the regulatory mechanism of NUDT15. Significant differences were found in genotype and allele frequencies among commercial broilers, commercial laying hens and dual-purpose chickens. The results of association analyses showed that these two indel loci could significantly affect growth traits, such as body weight, and carcass traits. Tissue expression profiling at E12 showed that the expression of NUDT15 was significantly higher in skeletal muscle, and time-expression profiling of leg muscle showed that the expression of NUDT15 in myoblasts was significantly higher in the E10 and E12 proliferation stages than in other stages. Promoter activity analysis showed that pro-1673-I and pro-1673-D significantly inhibited promoter activity, and the promoter activity of pro-1673-D was significantly lower than that of pro-1673-I. In addition, when NUDT15 was overexpressed or underwent interference in chicken primary myoblasts (CPMs), NUDT15 could inhibit the proliferation of CPMs. CONCLUSION: The results suggest that the studied indels in the promoter region of NUDT15 may regulate the proliferation of CPMs by affecting NUDT15 expression, ultimately affecting the growth and carcass traits of chickens. These indel polymorphisms may be used together as molecular markers for improving economic traits in chickens.

The Chicken Pan-Genome Reveals Gene Content Variation and a Promoter Region Deletion in IGF2BP1 Affecting Body Size.

Domestication and breeding have reshaped the genomic architecture of chicken, but the retention and loss of genomic elements during these evolutionary processes remain unclear. We present the first chicken pan-genome constructed using 664 individuals, which identified an additional approximately 66.5-Mb sequences that are absent from the reference genome (GRCg6a). The constructed pan-genome encoded 20,491 predicated protein-coding genes, of which higher expression levels are observed in conserved genes relative to dispensable genes. Presence/absence variation (PAV) analyses demonstrated that gene PAV in chicken was shaped by selection, genetic drift, and hybridization. PAV-based genome-wide association studies identified numerous candidate mutations related to growth, carcass composition, meat quality, or physiological traits. Among them, a deletion in the promoter region of IGF2BP1 affecting chicken body size is reported, which is supported by functional studies and extra samples. This is the first time to report the causal variant of chicken body size quantitative trait locus located at chromosome 27 which was repeatedly reported. Therefore, the chicken pan-genome is a useful resource for biological discovery and breeding. It improves our understanding of chicken genome diversity and provides materials to unveil the evolution history of chicken domestication.

Genome-wide identification evolution and expression of vestigial-like gene family in chicken.

Vestigial-like (Vgll) genes are widespread in vertebrates and play an important role in muscle development. In this study, we used bioinformatics methods to systematically identify the chicken VGLL family in the whole genome and investigated its evolutionary history and gene structure features. Tissue expression spectra combined with real-time PCR data were used to analyze the organizational expression pattern of the genes. Based on the maximum likelihood method, a phylogenetic tree of the VGLL family was constructed, and 94 VGLL genes were identified in 24 breeds, among which four VGLL family genes were identified in the chicken genome. Ten motifs were detected in the VGLL genes, and the analysis of introns combined with gene structure revealed that the family was conserved during evolution. Tissue expression analysis suggested that the expression profiles of the VGLL family genes in 16 tissues differed between LU Shi and AA broilers. In addition, a single gene (VGLL2) showed increased expression in chickens at embryonic days 10-16 and was involved in the growth and development of skeletal muscle in chickens in the embryonic stage. In summary, VGLL genes are involved in chicken muscle growth and development, which provides useful information for subsequent functional studies of VGLL genes.

LncRNAs and their regulatory networks in breast muscle tissue of Chinese Gushi chickens during late postnatal development.

BACKGROUND: Chicken skeletal muscle is an important economic product. The late stages of chicken development constitute the main period that affects meat production. LncRNAs play important roles in controlling the epigenetic process of growth and development. However, studies on the role of lncRNAs in the late stages of chicken breast muscle development are still lacking. In this study, to investigate the expression characteristics of lncRNAs during chicken muscle development, 12 cDNA libraries were constructed from Gushi chicken breast muscle samples from 6-, 14-, 22-, and 30-week-old chickens. RESULTS: A total of 1252 new lncRNAs and 1376 annotated lncRNAs were identified. Furthermore, 53, 61, 50, 153, 117, and 78 DE-lncRNAs were found in the W14 vs. W6, W22 vs. W14, W22 vs. W6, W30 vs. W6, W30 vs. W14, and W30 vs. W22 comparison groups, respectively. After GO enrichment analysis of the DE-lncRNAs, several muscle development-related GO terms were found in the W22 vs. W14 comparison group. Moreover, it was found that the MAPK signaling pathway was one of the most frequently enriched pathways in the different comparison groups. In addition, 12 common target DE-miRNAs of DE-lncRNAs were found in different comparison groups, some of which were muscle-specific miRNAs, such as gga-miR-206, gga-miR-1a-3p, and miR-133a-3p. Interestingly, the precursors of four newly identified miRNAs were found to be homologous to lncRNAs. Additionally, we found some ceRNA networks associated with muscle development-related GO terms. For example, the ceRNA networks contained the DYNLL2 gene with 12 lncRNAs that targeted 2 miRNAs. We also constructed PPI networks, such as IGF-I-EGF and FZD6-WNT11. CONCLUSIONS: This study revealed, for the first time, the dynamic changes in lncRNA expression in Gushi chicken breast muscle at different periods and revealed that the MAPK signaling pathway plays a vital role in muscle development. Furthermore, MEF2C and its target lncRNA may be involved in muscle regulation through the MAPK signaling pathway. This research provided valuable resources for elucidating posttranscriptional regulatory mechanisms to promote the development of chicken breast muscles after hatching.

Global investigation of estrogen-responsive genes regulating lipid metabolism in the liver of laying hens.

BACKGROUND: Estrogen plays an essential role in female development and reproductive function. In chickens, estrogen is critical for lipid metabolism in the liver. The regulatory molecular network of estrogen in chicken liver is poorly understood. To identify estrogen-responsive genes and estrogen functional sites on a genome-wide scale, we determined expression profiles of mRNAs, lncRNAs, and miRNAs in estrogen-treated ((17ß-estradiol)) and control chicken livers using RNA-Sequencing (RNA-Seq) and studied the estrogen receptor α binding sites by ChIP-Sequencing (ChIP-Seq). RESULTS: We identified a total of 990 estrogen-responsive genes, including 962 protein-coding genes, 11 miRNAs, and 17 lncRNAs. Functional enrichment analyses showed that the estrogen-responsive genes were highly enriched in lipid metabolism and biological processes. Integrated analysis of the data of RNA-Seq and ChIP-Seq, identified 191 genes directly targeted by estrogen, including 185 protein-coding genes, 4 miRNAs, and 2 lncRNAs. In vivo and in vitro experiments showed that estrogen decreased the mRNA expression of PPARGC1B, which had been reported to be linked with lipid metabolism, by directly increasing the expression of miR-144-3p. CONCLUSIONS: These results increase our understanding of the functional network of estrogen in chicken liver and also reveal aspects of the molecular mechanism of estrogen-related lipid metabolism.

Genome-wide association study reveals the genetic determinism of growth traits in a Gushi-Anka F2 chicken population.

Chicken growth traits are economically important, but the relevant genetic mechanisms have not yet been elucidated. Herein, we performed a genome-wide association study to identify the variants associated with growth traits. In total, 860 chickens from a Gushi-Anka F2 resource population were phenotyped for 68 growth and carcass traits, and 768 samples were genotyped based on the genotyping-by-sequencing (GBS) method. Finally, 734 chickens and 321,314 SNPs remained after quality control and removal of the sex chromosomes, and these data were used to carry out a GWAS analysis. A total of 470 significant single-nucleotide polymorphisms (SNPs) for 43 of the 68 traits were detected and mapped on chromosomes (Chr) 1-6, -9, -10, -16, -18, -23, and -27. Of these, the significant SNPs in Chr1, -4, and -27 were found to be associated with more than 10 traits. Multiple traits shared significant SNPs, indicating that the same mutation in the region might have a large effect on multiple growth or carcass traits. Haplotype analysis revealed that SNPs within the candidate region of Chr1 presented a mosaic pattern. The significant SNPs and pathway enrichment analysis revealed that the MLNR, MED4, CAB39L, LDB2, and IGF2BP1 genes could be putative candidate genes for growth and carcass traits. The findings of this study improve our understanding of the genetic mechanisms regulating chicken growth and carcass traits and provide a theoretical basis for chicken breeding programs.

Effects of miR-125b-5p on Preadipocyte Proliferation and Differentiation in Chicken.

Our previous studies have shown that miR-125b-5p was highly expressed and significantly upregulated during abdominal fat deposition in chickens. However, the role of miR-125b in the regulation of adipogenesis is not clear in chickens. Therefore, we evaluated the effects of miR-125b-5p on preadipocyte proliferation and differentiation and the interaction between miR-125b-5p and the acyl-CoA synthetase bubblegum family member 2 (ACSBG2) gene in adipogenesis in chicken abdominal adipose tissue. Here, transfection tests of miR-125b-5p mimic/inhibitor were performed in preadipocytes, and the effects of miR-125b-5p on preadipocytes proliferation and differentiation were analyzed. The target site of miR-125b-5p in the 3'UTR (untranslated region) of ACSBG2 were verified by a luciferase reporter assay. Our results showed that miR-125b-5p overexpression inhibited proliferation and reduced the number of cells in S phase and G2/M phase in preadipocytes; conversely, miR-125b-5p inhibition promoted the proliferation and increased the number of cells in S phase and G2/M phase. In adipocytes after induction, miR-125b-5p overexpression led to a notable increase in the accumulation of lipid droplets as well as in the concentration of triglycerides, while miR-125b-5p inhibition had the opposite effect. Furthermore, miR-125b-5p could directly bind to the 3'UTR of ACSBG2, and its overexpression could significantly repress the mRNA and protein expression of ACSBG2. These results indicate that miR-125b-5p can inhibit preadipocyte proliferation and can promote preadipocyte differentiation to affect adipogenesis in chicken abdominal adipose tissues, at least partially by downregulating ACSBG2.

Breeding history and candidate genes responsible for black skin of Xichuan black-bone chicken.

BACKGROUND: Domesticated chickens have a wide variety of phenotypes, in contrast with their wild progenitors. Unlike other chicken breeds, Xichuan black-bone chickens have blue-shelled eggs, and black meat, beaks, skin, bones, and legs. The breeding history and the economically important traits of this breed have not yet been explored at the genomic level. We therefore used whole genome resequencing to analyze the breeding history of the Xichuan black-bone chickens and to identify genes responsible for its unique phenotype. RESULTS: Principal component and population structure analysis showed that Xichuan black-bone chicken is in a distinct clade apart from eight other breeds. Linkage disequilibrium analysis showed that the selection intensity of Xichuan black-bone chickens is higher than for other chicken breeds. The estimated time of divergence between the Xichuan black-bone chickens and other breeds is 2.89 ka years ago. Fst analysis identified a selective sweep that contains genes related to melanogenesis. This region is probably associated with the black skin of the Xichuan black-bone chickens and may be the product of long-term artificial selection. A combined analysis of genomic and transcriptomic data suggests that the candidate gene related to the black-bone trait, EDN3, might interact with the upstream ncRNA LOC101747896 to generate black skin color during melanogenesis. CONCLUSIONS: These findings help explain the unique genetic and phenotypic characteristics of Xichuan black-bone chickens, and provide basic research data for studying melanin deposition in animals.

Detection of CNV in the SH3RF2 gene and its effects on growth and carcass traits in chickens.

BACKGROUND: The SH3RF2 gene is a protein-coding gene located in a quantitative trait locus associated with body weight, and its deletion has been shown to be positively associated with body weight in chickens. RESULTS: In the present study, CNV in the SH3RF2 gene was detected in 4079 individuals from 17 populations, including the "Gushi ×Anka" F2 resource population and populations of Chinese native chickens, commercial layers, and commercial broilers. The F2 resource population was then used to investigate the genetic effects of the chicken SH3RF2 gene. The results showed that the local chickens and commercial layers were all homozygous for the wild-type allele. Deletion mutation individuals were detected in all of the commercial broiler breeds except Hubbard broiler. A total of, 798 individuals in the F2 resource group were used to analyze the effects of genotype (DD/ID/II) on chicken production traits. The results showed that CNV was associated with 2-, 6-, 10-, and 12-week body weight (P = 0.026, 0.042, 0.021 and 0.039 respectively) and significantly associated with 8-week breast bone length (P = 0.045). The mutation was significantly associated with 8-week body weight (P = 0.007) and 4-week breast bone length (P = 0.010). CNV was significantly associated with evisceration weight, leg muscle weight, carcass weight, breast muscle weight and gizzard weight (P = 0.032, 0.033, 0.045, 0.004 and 0.000, respectively). CONCLUSIONS: CNV of the SH3RF2 gene contributed to variation in the growth and weight gain of chickens.